| 1. |
- del Peso-Santos, Teresa, et al.
(författare)
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Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2.
- 2006
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Ingår i: J Bacteriol. - 0021-9193. ; 188:13, s. 4812-21
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Tidskriftsartikel (refereegranskat)abstract
- The P(styA) promoter of Pseudomonas sp. strain Y2 controls expression of the styABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of two paa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of the paa gene clusters of this strain. In silico analysis of the P(styA) promoter region revealed a sequence located just within styA that is similar to the reported PaaX binding sites of Escherichia coli and the proposed PaaX binding sites of the paa genes of Pseudomonas species. Here we show that protein extracts from some Pseudomonas strains that have paaX genes, but not from a paaX mutant strain, can bind and retard the migration of a P(styA) specific probe. Purified maltose-binding protein (MBP)-PaaX1 fusion protein specifically binds the P(styA) promoter proximal PaaX site, and this binding is eliminated by the addition of phenylacetyl-coenzyme A. The sequence protected by MBP-PaaX1 binding was defined by DNase I footprinting. Moreover, MBP-PaaX1 represses transcription from the P(styA) promoter in a phenylacetyl-coenzyme A-dependent manner in vitro. Finally, the inactivation of both paaX gene copies of Pseudomonas sp. strain Y2 leads to a higher level of transcription from the P(styA) promoter, while heterologous expression of the PaaX1 in E. coli greatly decreases transcription from the P(styA) promoter. These findings reveal a control mechanism that integrates regulation of styrene catabolism by coordinating the expression of the styrene upper catabolic operon to that of the paa-encoded central pathway and support a role for PaaX as a major regulatory protein in the phenylacetyl-coenzyme A catabolon through its response to the levels of this central metabolite.
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| 2. |
- Sodergren, Erica, et al.
(författare)
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The genome of the sea urchin Strongylocentrotus purpuratus.
- 2006
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 314:5801, s. 941-52
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Tidskriftsartikel (refereegranskat)abstract
- We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.
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| 3. |
- Vaz-Dominguez, Cristina, et al.
(författare)
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Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition
- 2008
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 24, s. 531-537
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Tidskriftsartikel (refereegranskat)abstract
- Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochem. modified with Ph derivs. as a way to attach the enzyme mols. with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5 mA/cm2 of electrocatalytic redn. of O2 to H2O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu center of the laccase towards the electrode. The covalent attachment of the laccase mols. to the functionalized electrodes permitted remarkable operational stability. Moreover, O2 bioelectroredn. based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic redn. of O2. Thus, two different modes of laccase inhibition by halides are discussed.
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| 4. |
- Zumarraga, Miren, et al.
(författare)
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Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant : the connection between the C-terminal plug and the conserved 509VSG511 tripeptide
- 2008
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Ingår i: Combinatorial chemistry & high throughput screening. - : Bentham Science Publishers Ltd.. - 1386-2073 .- 1875-5402. ; 11:10, s. 807-816
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Tidskriftsartikel (refereegranskat)abstract
- A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial satn. mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The KmO2 value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the 509VSG511 tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.
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